Modulation of forkhead box O1A expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of forkhead box O1A. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding forkhead box O1A. Methods of using these compounds for modulation of forkhead box O1A expression and for treatment of diseases associated with expression of forkhead box O1A are provided, in particular, for methods of treating diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/860,137 filed Aug. 20, 2010, which is a divisional of U.S.application Ser. No. 11/739,017 filed Apr. 23, 2007, issued as U.S. Pat.No. 7,807,649 on Oct. 5, 2010, which is a divisional of U.S. applicationSer. No. 10/671,074 filed Sep. 25, 2003, issued as U.S. Pat. No.7,229,976 on Jun. 12, 2007, which is a continuation-in-part of U.S.application Ser. No. 10/260,203 filed Sep. 26, 2002, now abandoned, eachof which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledHTS0008USC1SEQ.txt, created on Jun. 21, 2012 which is 60 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of forkhead box 01A. In particular, this inventionrelates to compounds, particularly oligonucleotide compounds which, insome embodiments, hybridize with nucleic acid molecules encodingforkhead box 01A. Such compounds are shown herein to modulate theexpression of forkhead box 01 A.

BACKGROUND OF THE INVENTION

The forkhead gene family, originally identified in Drosophila, encodes aclass of transcription factors that are important for embryogenesis anddevelopment. The forkhead domain, also referred to as the “winged helixdomain”, is a 100 amino acid sequence which forms a variation of ahelix-turn-helix such that the target DNA is recognized by analpha-helix and two large loops or “wings”. The action of transcriptionfactors is essential for proper development since vertebrate developmentrelies on appropriate temporal and spatial expression of genes.Consequently, errors in the action of forkhead transcription factors mayhelp to identify the molecular basis for developmental defects (Gajiwalaand Burley, Curro Opin. Struct. Biol., 2000, 10, 110-116).

One of these forkhead genes, forkhead box 01A (also called FOX01A,forkhead in rhabdomyosarcoma, forkhead box 01A (rhabdomyosarcoma),forkhead (drosophila) homolog 1, FKHR, FKH1, FKHR/PAX3 fusion gene, andFKHR/PAX7 fusion gene) was identified and cloned in 1993 based on achromosomal translocation that is frequently found to be overexpressedor amplified in a high proportion of alveolar rhabdomyosarcomas (ARMS)(Davis and Barr, Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 8047-8051;Davis et al., Cancer Res., 1994, 54, 2869-2872; Galili et al., Nat.Genet., 1993, 5, 230-235). Rhabdomyosarcomas are a group of malignanttumors which are the most common soft-tissue sarcoma of childhood, andARMS is associated with the expression of two fusion proteins: one is afusion of the transcription factor PAX3 to forkhead box O1A and thesecond is a fusion of the transcription factor PAX7 to forkhead box O1A.These fusion genes arise from the chromosomal translocation of the geneencoding the transcription factor PAX3, located on chromosome 2, or PAX7located on chromosome 1, to a position on chromosome 13 adjacent to theforkhead box O1A gene. Intron 1 of the forkhead box O1A gene isrearranged in t(2; 13)-containing alveolar rhabdomyosarcomas (Davis etal., Hum. Mol. Genet., 1995, 4, 2355-2362). The fusion protein resultingfrom the t(2; 13) translocation contains the N-terminal region of PAX3,including the DNA-binding domains, and the C-terminal transcriptionactivation domain of forkhead box O1A, with truncation of the forkheadDNA-binding domain (Anderson et al., Genes. Chromosomes Cancer, 1999,26, 275-285).

Diabetes and its complications are a serious problem for the populationsof industrialized countries. Generally, this disease results fromimpaired insulin production from pancreatic β-cells. In type 2 diabetes,a combination of genetic and environmental factors brings about β-cellfailure, which results in impaired insulin secretion and activity. Incontrast, an autoimmune process destroys β-cells in type 1 diabetes.Since most individuals with type 2 diabetes are insulin resistant, it iscommonly thought that the n-cells failure observed in individuals withtype 2 diabetes is related to insulin resistance.

The forkhead box O1A protein may play a role in the progression ofdiabetes as well as several other diseases. Forkhead box O1A is thetranscription factor that binds the insulin response element in theinsulin-like growth factor binding protein-1 (IGF-BP-1) promoter (Durhamet al., Endocrinology, 1999, 140, 3140-3146). It has been observed thatinsulin also regulates the activity of forkhead box O1A as atranscription factor of glucose-6-phosphatase, a key enzyme ingluconeogenesis (Nakae et al., J. Clin. Invest., 2001, 108, 1359-1367).Nakae et al. showed that Foxol controls two important processes in thepathogenesis of type 2 diabetes: hepatic glucose production and β-cellcompensation of insulin resistance. The data suggest a common mechanismby which insulin resistance would bring about metabolic alterations thatcause type 2 diabetes. Furthermore, Guo et al. (J. Biol. Chem., 1999,274, 17184-17192) determined that forkhead box O1A stimulates promoteractivity through an insulin response sequence. In addition, Guo et al.,using a mutant forkhead box O1A, in which phosphorylation sites weremutated to prevent phosphorylation, showed that phosphorylation offorkhead box O1A by protein kinase B is necessary in order for insulinto disrupt transcription of target genes by forkhead box O1A. It hasfurther been shown that forkhead box O1A activity, but not thePAX3/forkhead box O1A fusion protein activity, can also be suppressed byphosphorylation by Akt (del Peso et al., Oncogene, 1999, 18, 7328-7333;Tang et al., J. Biol. Chem., 1999, 274, 16741-16746). From theseobservations, it appears that forkhead box O1A may contribute to hepaticproduction of IGFBP-1 and unrestrained gluconeogenesis in Type 2diabetes because insulin is not able to regulate the activity offorkhead box O1 as a transcription factor of IGFBP-1.

Prior to the present invention, there were no known therapeutic agentsthat effectively inhibited the synthesis of forkhead box O1A.

The present invention provides compositions and methods for modulatingforkhead box O1A expression.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, particularly nucleicacid and nucleic acid-like oligomers, which are targeted to a nucleicacid encoding forkhead box O1A, and which modulate the expression offorkhead box O1A. Pharmaceutical and other compositions comprising thecompounds of the invention are also provided. Also provided are methodsof screening for modulators of forkhead box O1A and methods ofmodulating the expression of forkhead box O1A in cells, tissues, oranimals comprising contacting the cells, tissues, or animals with one ormore of the compounds or compositions of the invention. Methods oftreating an animal, particularly a human, suspected of having or beingprone to a disease or condition associated with expression of forkheadbox O1A are also set forth herein. In particular, the compounds of thepresent invention are effective for treating diabetes, in particulartype 2 diabetes. Such methods comprise administering a therapeuticallyor prophylactically effective amount of one or more of the compounds orcompositions of the invention to the animal or human in need oftreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of an antisense compound in a glucose tolerancetest in lean C57B16 mice. Compound 188764 significantly improved glucosetolerance tests in these animals.

FIG. 2 shows the effects of an antisense compound on insulin levels inlean C57B16 mice. Compound 188764 decreased insulin levels by more than50% at the highest dose.

FIG. 3 shows the effects of an antisense compound on blood glucose aftera 16 hour fast in 9-12 week old db/db mice. Compound 188764significantly reduced blood glucose levels after a 16 hour fast in 9-12weeks db/db mice.

FIG. 4 shows the effects of an antisense compound on plasma insulinlevels in high fat diet C57B16 mice. Compound 188764 and rosiglitazonedecreased plasma insulin levels in high fat diet fed mice.

FIG. 5 shows effects of an antisense compound in a glucose tolerancetest in high fat fed C57B16 mice. Compound 188764 normalized insulinlevels in these animals.

DESCRIPTION OF EMBODIMENTS

The present invention employs compounds, such as oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding forkhead box O1A. In particular, the presentinvention provides compounds that are effective in treating diabetes, inparticular type 2 diabetes, as is demonstrated in the Examples below.

The present invention demonstrates that reducing forkhead levels is aneffective method for treating diabetes in a subject, particularly type 2diabetes. The present invention also demonstrates that reducing forkheadlevels is an effective method for decreasing blood or plasma glucose,improving glucose tolerance, and normalizing insulin levels. Any of thecompounds or compositions described herein can be administered to asubject, such as a human, in an amount effective to reduce forkheadlevels. Administration of compounds and/or compositions can be carriedout as described below. Such an amount can depend on, for example, thespecies of animal, weight, height, age, sex, stage of disease orcondition, and the like. Forkhead box O1A expression can be measuredand/or monitored by any of the methods described herein. The subjectscan be previously diagnosed with diabetes or can be susceptible todeveloping diabetes or at high risk of developing diabetes.

Forkhead box O1A controls two important processes in the pathogenesis oftype 2 diabetes: hepatic glucose production and β-cell compensation ofinsulin resistance. Forkhead box O1A has also been demonstrated tostimulate promoter activity through an insulin response sequence.Indeed, phosphorylation of forkhead box O1A by protein kinase B isnecessary for insulin to disrupt transcription of target genes byforkhead box O1A. Thus, it appears that forkhead box O1A may contributeto hepatic production of IGFBP-1 and unrestrained gluconeogenesis inType 2 diabetes because insulin is not able to regulate the activity offorkhead box O1 as a transcription factor of IGFBP-1.

The present invention employs compounds, such as oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding forkhead box O1A, which can be useful inpreventing or treating a hyperproliferative disorder, such as cancer,particularly, rhabdomyosarcoma. Expression of the PAX3/forkhead box O1Afusion protein is able to induce phenotypic changes, includingtransforming fibroblasts in culture (Scheidler et al., Proc. Natl. Acad.Sci. U.S.A., 1996, 93, 9805-9809) and inducing the morphologicalfeatures of ARMS in vivo (Anderson et al., Am. J. Pathol., 2001, 159,1089-1096). The PAX3/forkhead box O1A fusion protein confers uponembryonal rhabdomyosarcoma cells protection from apoptosis, a keyfeature of tumorigenicity, at least in part by transcriptionalregulation of the anti-apoptotic protein BCL-XL (Bernasconi et al.,Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 13164-13169; Margue et al.,Oncogene, 2000, 19, 2921-2929). The PAX3/forkhead box O1A fusion proteintranscribes genes normally transcribed by PAX3 and may even transcribegenes that are not targets of PAX3, as is the case with theplatelet-derived growth factor alpha receptor promoter (Epstein et al.,Mol. Cell. Biol., 1998, 18, 4118-4130). The PAX3/forkhead box O1A fusionprotein is a more potent transcriptional activator than the normal PAX3protein, and it has been suggested that this upregulation of genes thatare normally targets of PAX3 could be a critical event in the histologyof ARMS (Bennicelli et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93,5455-5459). Expression of the PAX3/forkhead box O1A fusion protein mayalso interfere with normal developmental functions of PAX3 as evidencedin transgenic mice (Anderson et al., Proc. Natl. Acad. Sci. U.S.A.,2001, 98, 1589-1594).

The present invention provides compounds that reduce the level of bothhuman and murine forkhead box O1A. In addition, the compounds describedherein can also reduce the level of monkey forkhead box O1A. This isdemonstrated by both in vitro and in vivo assays as set forth in theExamples below. In addition, it is shown that reducing forkhead levelshas beneficial effects on various animal models of diabetes, normalizingglucose intolerance and insulin levels, and decreasing blood glucoselevels. This is accomplished by providing oligonucleotides or otheroligomeric compounds that specifically hybridize with one or morenucleic acid molecules encoding forkhead box O1A. As used herein, theterms “target nucleic acid” and “nucleic acid molecule encoding forkheadbox O1A” have been used for convenience to encompass DNA encodingforkhead box O1A, RNA (including pre-mRNA and mRNA, or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of a compound of this invention with its target nucleicacid is generally referred to as “antisense”. Consequently, a mechanismbelieved to be included in the practice of some embodiments of theinvention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, specific nucleic acid molecules and theirfunctions for such antisense inhibition are targeted.

Functions of DNA to be interfered with can include, but are not limitedto, replication and transcription. Replication and transcription, forexample, can be from an endogenous cellular template, a vector, aplasmid construct or otherwise. The functions of RNA to be interferedwith can include, but are not limited to, functions such astranslocation of the RNA to a site of protein translation, translocationof the RNA to sites within the cell which are distant from the site ofRNA synthesis, translation of protein from the RNA, splicing of the RNAto yield one or more RNA species, and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. One result of such interference with target nucleic acidfunction is modulation of the expression of forkhead box O1A. In thecontext of the present invention, “modulation” and “modulation ofexpression” mean either an increase (stimulation) or a decrease(inhibition) in the amount or levels of a nucleic acid molecule encodingthe gene, e.g., DNA or RNA. Inhibition is one form of modulation ofexpression and mRNA is often a target nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,one mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays. Of course, the compounds of the invention neednot be specifically hybridizable to modulate the expression of forkheadbox O1A. Indeed, in some embodiments, the compounds of the invention arehybridizable to nucleic acid molecules encoding forkhead box O1A. Inother embodiments, the compounds are specifically hybridizable tonucleic acid molecules encoding forkhead box O1A.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomer or oligonucleotide mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). In some embodiments, the compounds ofthe present invention comprise at least 70%, or at least 75%, or atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%sequence complementarity to a target region within the target nucleicacid. For example, an antisense compound in which 18 of 20 nucleobasesof the antisense compound are complementary to a target region, andwould therefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases that are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, that uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, homology, sequence identity or complementarity,between the oligomeric and target is between about 50% to about 60%. Insome embodiments, homology, sequence identity or complementarity, isbetween about 60% to about 70%. In other embodiments, homology, sequenceidentity or complementarity, is between about 70% and about 80%. Inother embodiments, homology, sequence identity or complementarity, isbetween about 80% and about 90%. In other embodiments, homology,sequence identity or complementarity, is about 90%, about 92%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

According to the present invention, compounds include, but are notlimited to, antisense oligomeric compounds, antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, alternatesplicers, primers, probes, and other oligomeric compounds whichhybridize to at least a portion of the target nucleic acid. As such,these compounds may be introduced in the form of single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges orloops. Once introduced to a system, the compounds of the invention mayelicit the action of one or more enzymes or structural proteins toeffect modification of the target nucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While one form of antisense compound is a single-stranded antisenseoligonucleotide, in many species the introduction of double-strandedstructures, such as double-stranded RNA (dsRNA) molecules, has beenshown to induce potent and specific antisense-mediated reduction of thefunction of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown thatthe primary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507).The posttranscriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it hasbeen shown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 2002, 295, 694-697).

The oligonucleotides and oligomers of the present invention also includevariants in which a different base is present at one or more of thenucleotide positions in the oligonucleotide or oligomer. For example, ifthe first nucleotide is an adenosine, variants may be produced whichcontain thymidine, guanosine or cytidine at this position. This may bedone at any of the positions of the oligonucleotide. Theseoligonucleotides are then tested using the methods described herein todetermine their ability to inhibit expression of forkhead box O1A mRNA.

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions that functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are one form of the compounds of this invention,the present invention comprehends other families of compounds as well,including but not limited to, oligonucleotide analogs and mimetics suchas those described herein.

The compounds in accordance with this invention comprise from about 8 toabout 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).One of ordinary skill in the art will appreciate that the inventionembodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, or 80 nucleobases in length.

In one embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another embodiment, the compounds of the invention are 15 to 30nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Antisense compounds 8 to 80 nucleobases in length comprising a stretchof at least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary antisense compounds include oligonucleotide sequences thatcomprise at least the 8 consecutive nucleobases from the 5′-terminus ofone of the illustrative antisense compounds (the remaining nucleobasesbeing a consecutive stretch of the same oligonucleotide beginningimmediately upstream of the 5′-terminus of the antisense compound whichis specifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases).Similarly, antisense compounds can be represented by oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the3′-terminus of one of the illustrative antisense compounds (theremaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately downstream of the 3′-terminus ofthe antisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). One having skill in the art armed with theantisense compounds illustrated herein will be able, without undueexperimentation, to identify additional antisense compounds.

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes forkhead box O1A.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that a desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be utilized fortranslation initiation in a particular cell type or tissue, or under aparticular set of conditions. In the context of the invention, “startcodon” and “translation initiation codon” refer to the codon or codonsthat are used in vivo to initiate translation of an mRNA transcribedfrom a gene encoding forkhead box O1A, regardless of the sequence(s) ofsuch codons. It is also known in the art that a translation terminationcodon (or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, oneregion is the intragenic region encompassing the translation initiationor termination codon of the open reading frame (ORF) of a gene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. The 5′ cap regioncan also be targeted.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also target sites. mRNA transcripts produced via the process ofsplicing of two (or more) mRNAs from different gene sources are known as“fusion transcripts.” It is also known that introns can be effectivelytargeted using antisense compounds targeted to, for example, DNA orpre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants.” More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants.”Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant produces a unique mRNA variant as a result ofsplicing. These mRNA variants are also known as “alternative splicevariants.” If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are hereinbelow referred to as “target segments.” Asused herein, the term “target segment” is defined as at least an8-nucleobase portion of a target region to which an active antisensecompound is targeted. While not wishing to be bound by theory, it ispresently believed that these target segments represent portions of thetarget nucleic acid which are accessible for hybridization.

While the specific sequences of certain target segments are set forthherein, one of skill in the art will recognize that these serve toillustrate and describe particular embodiments within the scope of thepresent invention. Additional target segments may be identified by onehaving ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative target segments are considered to be suitable for targetingas well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlytarget segments are represented by DNA or RNA sequences that comprise atleast the 8 consecutive nucleobases from the 3′-terminus of one of theillustrative target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). One havingskill in the art armed with the target segments illustrated herein willbe able, without undue experimentation, to identify additional targetsegments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

The oligomeric compounds are also targeted to or not targeted to regionsof the target nucleobase sequence (e.g., such as those disclosed inExample 13) comprising nucleobases 1-50, 51-100, 101-150, 151-200,201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600,601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000,1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300,1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600,1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900,1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200,2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500,2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800,2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100,3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400,3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3751-3700,3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000,4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300,4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600,4601-4650, 4751-4700, 4701-4750, or 4751-4800, 4801-4850, 4851-4900,4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200,5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500,5501-5550, 5551-5600, 5601-5650, 5751-5700, 5701-5723, or anycombination thereof.

In a further embodiment, the “target segments” identified herein may beemployed in a screen for additional compounds that modulate theexpression of forkhead box O1A. “Modulators” are those compounds thatdecrease or increase the expression of a nucleic acid molecule encodingforkhead box O1A and which comprise at least an 8-nucleobase portionwhich is complementary to a target segment. The screening methodcomprises the steps of contacting a target segment of a nucleic acidmolecule encoding forkhead box O1A with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodingforkhead box O1A. Once it is shown that the candidate modulator ormodulators are capable of modulating (e.g. either decreasing orincreasing) the expression of a nucleic acid molecule encoding forkheadbox O1A, the modulator may then be employed in further investigativestudies of the function of forkhead box O1A, or for use as a research,diagnostic, or therapeutic agent in accordance with the presentinvention.

The target segments of the present invention may be also be combinedwith their respective complementary antisense compounds of the presentinvention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocesssing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and target segments identifiedherein in drug discovery efforts to elucidate relationships that existbetween forkhead box O1A and a disease state, phenotype, or condition.These methods include detecting or modulating forkhead box O1Acomprising contacting a sample, tissue, cell, or organism with thecompounds of the present invention, measuring the nucleic acid orprotein level of forkhead box O1A and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther compound of the invention. These methods can also be performedin parallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

The compounds of the present invention can be utilized for diagnostics,therapeutics, prophylaxis and as research reagents and kits.Furthermore, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds of the invention are also useful for research anddiagnostics because, for example, these compounds hybridize to nucleicacids encoding forkhead box O1A. For example, oligonucleotides that areshown to hybridize with such efficiency and under such conditions asdisclosed herein as to be effective forkhead box O1A inhibitors willalso be effective primers or probes under conditions favoring geneamplification or detection, respectively. These primers and probes areuseful in methods requiring the specific detection of nucleic acidmolecules encoding forkhead box O1A and in the amplification of thenucleic acid molecules for detection or for use in further studies offorkhead box O1A. Hybridization of the antisense oligonucleotides,particularly the primers and probes, of the invention with a nucleicacid encoding forkhead box O1A can be detected by means known in theart. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of forkhead box O1A in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues, and animals, especially humans.

For therapeutics, an animal, such as a human, suspected of having adisease or disorder which can be treated by modulating the expression offorkhead box O1A, is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of a forkheadbox O1A inhibitor. The forkhead box O1A inhibitors of the presentinvention effectively inhibit the activity of the forkhead box O1Aprotein or inhibit the expression of the forkhead box O1A protein. Inone embodiment, the activity or expression of forkhead box O1A in ananimal is inhibited or decreased by about 10%. In other embodiments, theactivity or expression of forkhead box O1A in an animal is inhibited ordecreased by about 30%, or by 50% or more. Thus, the oligomericcompounds modulate expression of forkhead box O1A mRNA by at least 10%,by at least 20%, by at least 25%, by at least 30%, by at least 40%, byat least 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of forkhead box O1A may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal: In some embodiments, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding forkhead box O1A protein and/or the forkhead box O1Aprotein itself. Exemplary tissues include, but are not limited to,liver, fat, heart, lung, muscle, pancreas, spleen, testes, ovary, andsmall intestine.

The compounds of the invention can be utilized in compositions orpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Use of thecompounds and methods of the invention may also be usefulprophylactically.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound.Linear compounds, however, are generally more often used. In addition,linear compounds may have internal nucleobase complementarity and maytherefore fold in a manner as to produce a fully or partiallydouble-stranded compound. Within oligonucleotides, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Specific examples of antisense compounds useful in this inventioninclude, but are not limited to, oligonucleotides containing modifiedinternucleoside linkages (backbones) such as modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Modified oligonucleotide backbones containing a phosphorus atom thereininclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotri-esters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage (i.e., a single invertednucleoside residue which may be abasic—the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms may also be included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, each of which is herein incorporated by reference in itsentirety.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These include,but are not limited to, those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; riboacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis incorporated herein by reference in its entirety.

In other oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e., the backbone) of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference in its entirety. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

The compounds of the invention can include phosphorothioate backbonesand heteroatom backbones, such as, for example, —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— (known as a methylene (methylimino) or MMI backbone),—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—(wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—) of the above referenced U.S. Pat. No. 5,489,677, and theamide backbones of the above referenced U.S. Pat. No. 5,602,240. Alsoincluded are oligonucleotides having morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. In such cases, oligonucleotides may comprise one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Additional modifications include, butare not limited to, O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where nand m are from 1 to about 10. Other oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.One modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also knownas 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta,1995, 78, 486-504) (i.e., an alkoxyalkoxy group). Another modificationincludes 2′-dimethylaminooxyethoxy (i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE as described in examples hereinbelow), and2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE, i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow).

Other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. One 2′-arabinomodification is 2′-F. Similar modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotidesand the 5′ position of 5′ terminal nucleotide. Oligonucleotides may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative United States patents that teachthe preparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of whichis incorporated herein by reference in its entirety.

Another modification of the sugar includes Locked Nucleic Acids (LNAs)in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom ofthe sugar ring, thereby forming a bicyclic sugar moiety. The linkage canbe a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in WO 98/39352 and WO 99/14226.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Additional modified nucleobases include tricyclicpyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are suitable base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096;5,750,692 and 5,681,941, each of which is incorporated herein byreference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include, but arenot limited to, lipid moieties such as a cholesterol moiety, cholicacid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, analiphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis incorporated herein by reference in its entirety.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is incorporated herein by reference in its entirety.

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is incorporated herein byreference in its entirety.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention can be prepared as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed in WO 93/24510, WO 94/26764, and U.S. Pat. No. 5,770,713.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention(i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto). Foroligonucleotides, examples of pharmaceutically acceptable salts andtheir uses are further described in, for example, U.S. Pat. No.6,287,860, which is incorporated herein by reference in its entirety.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., J. of Pharma Sci., 1977, 66, 1-19). Thebase addition salts of said acidic compounds are prepared by contactingthe free acid form with a sufficient amount of the desired base toproduce the salt in the conventional manner. The free acid form may beregenerated by contacting the salt form with an acid and isolating thefree acid in the conventional manner. The free acid forms differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents, but otherwise the salts are equivalentto their respective free acid for purposes of the present invention. Asused herein, a “pharmaceutical addition salt” includes apharmaceutically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These include organicor inorganic acid salts of the amines. Examples of acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude, but are not limited to, (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The oligomeric compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, such as a human, suspected of havinga disease or disorder which can be treated by modulating the expressionof forkhead box O1A is treated by administering oligomeric compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan oligomeric compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the oligomeric compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The oligomeric compounds of the invention are useful for, inter alia,research and diagnostics, because these compounds hybridize to nucleicacids encoding forkhead box O1A, enabling sandwich and other assays toeasily be constructed to exploit this fact. Hybridization of theoligonucleotides of the invention with a nucleic acid encoding forkheadbox O1A can be detected by means known in the art. Such means mayinclude, but are not limited to, conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of forkhead box O1A in a sample may also be prepared.

The present invention also includes compositions, pharmaceuticalcompositions and formulations that include the oligomeric compounds ofthe invention. The pharmaceutical compositions of the present inventionmay be administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricularadministration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

Compositions, pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Topical formulations includethose in which the oligonucleotides of the invention are in admixturewith a topical delivery agent such as lipids, liposomes, fatty acids,fatty acid esters, steroids, chelating agents and surfactants. Lipidsand liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Fatty acids and esters include, but are not limited to,arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylicacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine,an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM),monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.Topical formulations are described in detail in U.S. patent applicationSer. No. 09/315,298 filed on May 20, 1999 which is incorporated hereinby reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Oral formulations are thosein which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Surfactants include fatty acids and/or esters or saltsthereof, bile acids and/or salts thereof. Bile acids/salts includechenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA),cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid,glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodiumglycodihydrofusidate. Fatty acids include arachidonic acid, undecanoicacid, oleic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Combinations of penetration enhancers include,for example, fatty acids/salts in combination with bile acids/salts. Onecombination is the sodium salt of lauric acid, capric acid and UDCA.Additional penetration enhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may bedelivered orally, in granular form including sprayed dried particles, orcomplexed to form micro or nanoparticles. Oligonucleotide complexingagents include poly-amino acids; polyimines; polyacrylates;polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationizedgelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) andstarches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,pollulans, celluloses and starches. Complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAF-albumin and DEAF-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999), each of whichis incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions may be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions may containadditional components in addition to the dispersed phases, and theactive drug that may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765). Variousliposomes comprising one or more glycolipids are known in the art.Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reportedthe ability of monosialoganglioside G_(M1), galactocerebroside sulfateand phosphatidylinositol to improve blood half-lives of liposomes. Thesefindings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci.U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, bothto Allen et al., disclose liposomes comprising (1) sphingomyelin and (2)the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat.No. 5,543,152 (Webb et al.) discloses liposomes comprisingsphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C1215G, thatcontains a PEG moiety. Iliumi et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates that are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid droplets thatare so highly deformable that they are easily able to penetrate throughpores which are smaller than the droplet. Transfersomes are adaptable tothe environment in which they are used, e.g. they are self-optimizing(adaptive to the shape of pores in the skin), self-repairing, frequentlyreach their targets without fragmenting, and often self-loading. To maketransfersomes it is possible to add surface edge-activators, usuallysurfactants, to a standard liposomal composition. Transfersomes havebeen used to deliver serum albumin to the skin. Thetransfersome-mediated delivery of serum albumin has been shown to be aseffective as subcutaneous injection of a solution containing serumalbumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935).Various natural bile salts, and their synthetic derivatives, act aspenetration enhancers. Thus the term “bile salts” includes any of thenaturally occurring components of bile as well as any of their syntheticderivatives. The bile salts of the invention include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption ofoligonucleotides through the mucosa is enhanced. With regards to theiruse as penetration enhancers in the present invention, chelating agentshave the added advantage of also serving as DNase inhibitors, as mostcharacterized DNA nucleases require a divalent metal ion for catalysisand are thus inhibited by chelating agents (Jarrett, J. Chromatogr.,1993, 618, 315-339). Chelating agents of the invention include but arenot limited to disodium ethylenediaminetetraacetate (EDTA), citric acid,salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of oligonucleotides through the alimentary mucosa(Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers include, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphor-amiditeswere purchased from commercial sources (e.g. Chemgenes, Needham Mass. orGlen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substitutednucleoside amidites are prepared as described in U.S. Pat. No.5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, optimized synthesis cycles weredeveloped that incorporate multiple steps coupling longer wait timesrelative to standard synthesis cycles.

The following abbreviations are used in the text: thin layerchromatography (TLC), melting point (MP), high pressure liquidchromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar),methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethylformamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO),tetrahydrofuran (THF).

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.) or prepared as follows:

Preparation of 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyldC amidite

To a 50 L glass reactor equipped with air stirrer and Ar gas line wasadded thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) atambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol,1.05 eq) was added as a solid in four portions over 1 h. After 30 min,TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent andby-products and 2% 3′,5′-bis DMT product (Rf in EtOAc 0.45, 0.05, 0.98,0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂ wereadded with stirring (pH of the aqueous layer 7.5). An additional 18 L ofwater was added, the mixture was stirred, the phases were separated, andthe organic layer was transferred to a second 50 L vessel. The aqueouslayer was extracted with additional CH₂Cl₂ (2×2 L). The combined 0.30organic layer was washed with water (10 L) and then concentrated in arotary evaporator to approx. 3.6 kg total weight. This was redissolvedin CH₂Cl₂ (3.5 L), added to the reactor followed by water (6 L) andhexanes (13 L). The mixture was vigorously stirred and seeded to give afine white suspended solid starting at the interface. After stirring for1 h, the suspension was removed by suction through a ½″ diameter teflontube into a 20 L suction flask, poured onto a 25 cm Coors Buchnerfunnel, washed with water (2×3 L) and a mixture of hexanes—CH₂Cl₂ (4:1,2×3 L) and allowed to air dry overnight in pans (1″ deep). This wasfurther dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constantweight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicateda trace contamination of the bis DMT product. NMR spectroscopy alsoindicated that 1-2 mole percent pyridine and about 5 mole percent ofhexanes was still present.

Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineintermediate for 5-methyl-dC amidite

To a 50 L Schott glass-lined steel reactor equipped with an electricstirrer, reagent addition pump (connected to an addition funnel),heating/cooling system, internal thermometer and an Ar gas line wasadded 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrousacetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture waschilled with stirring to −10° C. internal temperature (external −20°C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30minutes while maintaining the internal temperature below −5° C.,followed by a wash of anhydrous acetonitrile (1 L). Note: the reactionis mildly exothermic and copious hydrochloric acid fumes form over thecourse of the addition. The reaction was allowed to warm to 0° C. andthe reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; Rf 0.43to 0.84 of starting material and silyl product, respectively). Uponcompletion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60min so as to maintain the temperature between −20° C. and −10° C. duringthe strongly exothermic process, followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h. TLC indicated a complete conversion to the triazole product (Rf 0.83to 0.34 with the product spot glowing in long wavelength UV light). Thereaction mixture was a peach-colored thick suspension, which turneddarker red upon warming without apparent decomposition. The reaction wascooled to −15° C. internal temperature and water (5 L) was slowly addedat a rate to maintain the temperature below +10° C. in order to quenchthe reaction and to form a homogenous solution. (Caution: this reactionis initially very strongly exothermic). Approximately one-half of thereaction volume (22 L) was transferred by air pump to another vessel,diluted with EtOAc (12 L) and extracted with water (2×8 L). The combinedwater layers were back-extracted with EtOAc (6 L). The water layer wasdiscarded and the organic layers were concentrated in a 20 L rotaryevaporator to an oily foam. The foam was coevaporated with anhydrousacetonitrile (4 L) to remove EtOAc. (note: dioxane may be used insteadof anhydrous acetonitrile if dried to a hard foam). The second half ofthe reaction was treated in the same way. Each residue was dissolved indioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. Ahomogenous solution formed in a few minutes and the reaction was allowedto stand overnight (although the reaction is complete within 1 h).

TLC indicated a complete reaction (product Rf 0.35 in EtOAc-MeOH 4:1).The reaction solution was concentrated on a rotary evaporator to a densefoam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.),combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted withEtOAc (2 L). The organic layers were combined and concentrated to about8 kg total weight, cooled to 0° C. and seeded with crystalline product.After 24 hours, the first crop was collected on a 25 cm Coors Buchnerfunnel and washed repeatedly with EtOAc (3×3 L) until a white powder wasleft and then washed with ethyl ether (2×3 L). The solid was put in pans(1″ deep) and allowed to air dry overnight. The filtrate wasconcentrated to an oil, then redissolved in EtOAc (2 L), cooled andseeded as before. The second crop was collected and washed as before(with proportional solvents) and the filtrate was first extracted withwater (2×1 L) and then concentrated to an oil. The residue was dissolvedin EtOAc (1 L) and yielded a third crop which was treated as aboveexcept that more washing was required to remove a yellow oily layer.

After air-drying, the three crops were dried in a vacuum oven (50° C.,0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g,respectively) and combined to afford 2550 g (85%) of a white crystallineproduct (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity.The mother liquor still contained mostly product (as determined by TLC)and a small amount of triazole (as determined by NMR spectroscopy), bisDMT product and unidentified minor impurities. If desired, the motherliquor can be purified by silica gel chromatography using a gradient ofMeOH (0-25%) in EtOAc to further increase the yield.

Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidinepenultimate intermediate for 5-methyl dC amidite

Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a50 L glass reactor vessel equipped with an air stirrer and argon line.Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) wasadded and the reaction was stirred at ambient temperature for 8 h.TLC(CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; Rf 0.25) indicated approx. 92%complete reaction. An additional amount of benzoic anhydride (44 g, 0.19mol) was added. After a total of 18 h, TLC indicated approx. 96%reaction completion. The solution was diluted with EtOAc (20 L), TEA(1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixturewas extracted with water (15 L, then 2×10 L). The aqueous layer wasremoved (no back-extraction was needed) and the organic layer wasconcentrated in 2×20 L rotary evaporator flasks until a foam began toform. The residues were coevaporated with acetonitrile (1.5 L each) anddried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressureliquid chromatography (HPLC) revealed a contamination of 6.3% ofN4,3′-O-dibenzoyl product, but very little other impurities.

The product was purified by Biotage column chromatography (5 kg Biotage)prepared with 65:35:1 hexanes-EtOAc-TEA (4 L). The crude product (800g), dissolved in CH₂Cl₂ (2 L), was applied to the column. The column waswashed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solventmixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containingthe product were collected, and any fractions containing the product andimpurities were retained to be resubjected to column chromatography. Thecolumn was re-equilibrated with the original 65:35:1 solvent mixture (17kg). A second batch of crude product (840 g) was applied to the columnas before. The column was washed with the following solvent gradients:65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA (15kg). The column was reequilibrated as above, and a third batch of thecrude product (850 g) plus impure fractions recycled from the twoprevious columns (28 g) was purified following the procedure for thesecond batch. The fractions containing pure product combined andconcentrated on a 20 L rotary evaporator, co-evaporated withacetonitrile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constantweight of 2023 g (85%) of white foam and 20 g of slightly contaminatedproduct from the third run. HPLC indicated a purity of 99.8% with thebalance as the diBenzoyl product.

[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N₄-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite)

5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidine(998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (300 ml) at 50° C. under reduced pressure,then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (15 ml) was added and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (2.5 L) and water (600 ml), and extracted with hexane(3×3 L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (7.5 L) and hexane (6 L). The two layers wereseparated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L)and water (3×2 L), and the phases were separated. The organic layer wasdried (Na₂SO₄), filtered and rotary evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedto a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g anoff-white foam solid (96%).

2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides were synthesized as described previously(Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841) and U.S. Pat. No.5,670,633, herein incorporated by reference. The preparation of2′-fluoropyrimidines containing a 5-methyl substitution are described inU.S. Pat. No. 5,861,493. Briefly, the protected nucleosideN6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizingcommercially available 9-beta-D-arabinofuranosyl-adenine as startingmaterial and whereby the 2′-alpha-fluoro atom is introduced by aSN2-displacement of a 2′-beta-triflate group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate isobutyryl-arabinofuranosylguanosine. Alternatively,isobutyryl-arabinofuranosylguanosine was prepared as described by Rosset al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of theTPDS group was followed by protection of the hydroxyl group with THP togive isobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination ofT-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known asMOE amidites) are prepared as follows, or alternatively, as per themethods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine intermediate

2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol),tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12L three necked flask and heated to 130° C. (internal temp) atatmospheric pressure, under an argon atmosphere with stirring for 21 h.TLC indicated a complete reaction. The solvent was removed under reducedpressure until a sticky gum formed (50-85° C. bath temp and 100-11 mmHg) and the residue was redissolved in water (3 L) and heated to boilingfor 30 min in order the hydrolyze the borate esters. The water wasremoved under reduced pressure until a foam began to form and then theprocess was repeated. HPLC indicated about 77% product, 15% dimer (5′ ofproduct attached to 2′ of starting material) and unknown derivatives,and the balance was a single unresolved early eluting peak.

The gum was redissolved in brine (3 L), and the flask was rinsed withadditional brine (3 L). The combined aqueous solutions were extractedwith chloroform (20 L) in a heavier-than continuous extractor for 70 h.The chloroform layer was concentrated by rotary evaporation in a 20 Lflask to a sticky foam (2400 g). This was coevaporated with MeOH (400mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolved atwhich point the vacuum was lowered to about 0.5 atm. After 2.5 L ofdistillate was collected a precipitate began to form and the flask wasremoved from the rotary evaporator and stirred until the suspensionreached ambient temperature. EtOAc (2 L) was added and the slurry wasfiltered on a 25 cm table top Buchner funnel and the product was washedwith EtOAc (3×2 L). The bright white solid was air dried in pans for 24h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) toafford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

The brine layer in the 20 L continuous extractor was further extractedfor 72 h with recycled chloroform. The chloroform was concentrated to120 g of oil and this was combined with the mother liquor from the abovefiltration (225 g), dissolved in brine (250 mL) and extracted once withchloroform (250 mL). The brine solution was continuously extracted andthe product was crystallized as described above to afford an additional178 g of crystalline product containing about 2% of thymine. Thecombined yield was 1827 g (69.4%). HPLC indicated about 99.5% puritywith the balance being the dimer.

Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridinepenultimate intermediate

In a 50 L glass-lined steel reactor,2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol),lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile(15 L). The solution was stirred rapidly and chilled to −10° C.(internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g,5.21 mol) was added as a solid in one portion. The reaction was allowedto warm to −2° C. over 1 h. (Note: The reaction was monitored closely byTLC (EtOAc) to determine when to stop the reaction so as to not generatethe undesired bis-DMT substituted side product). The reaction wasallowed to warm from −2 to 3° C. over 25 min. then quenched by addingMeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L).The solution was transferred to a clear 50 L vessel with a bottomoutlet, vigorously stirred for 1 minute, and the layers separated. Theaqueous layer was removed and the organic layer was washed successivelywith 10% aqueous citric acid (8 L) and water (12 L). The product wasthen extracted into the aqueous phase by washing the toluene solutionwith aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueouslayer was overlayed with toluene (12 L) and solid citric acid (8 moles,1270 g) was added with vigorous stirring to lower the pH of the aqueouslayer to 5.5 and extract the product into the toluene. The organic layerwas washed with water (10 L) and TLC of the organic layer indicated atrace of DMT-O-Me, bis DMT and dimer DMT.

The toluene solution was applied to a silica gel column (6 L sinteredglass funnel containing approx. 2 kg of silica gel slurried with toluene(2 L) and TEA (25 mL)) and the fractions were eluted with toluene (12 L)and EtOAc (3×4 L) using vacuum applied to a filter flask placed belowthe column. The first EtOAc fraction containing both the desired productand impurities were resubjected to column chromatography as above. Theclean fractions were combined, rotary evaporated to a foam, coevaporatedwith acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40°C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) togive a true dry weight of 2803 g (96%). HPLC indicated that the productwas 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown,0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.

Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite)

5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine(1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solutionwas co-evaporated with toluene (200 ml) at 50° C. under reducedpressure, then cooled to room temperature and 2-cyanoethyltetaisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g,1.0 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (20 ml) was added and the solution was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (3.5 L) and water (600 ml) and extracted with hexane(3×3 L). The mixture was diluted with water (1.6 L) and extracted withthe mixture of toluene (12 L) and hexanes (9 L). The upper layer waswashed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organiclayer was dried (Na2SO4), filtered and evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedin a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of anoff-white foamy solid (95%).

Preparation of5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate

To a 50 L Schott glass-lined steel reactor equipped with an electricstirrer, reagent addition pump (connected to an addition funnel),heating/cooling system, internal thermometer and argon gas line wasadded 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616kg, 4.23 mol, purified by base extraction only and no scrub column),anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). Themixture was chilled with stirring to −10° C. internal temperature(external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq)was added over 30 min. while maintaining the internal temperature below−5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: thereaction is mildly exothermic and copious hydrochloric acid fumes formover the course of the addition). The reaction was allowed to warm to 0°C. and the reaction progress was confirmed by TLC (EtOAc, Rf 0.68 and0.87 for starting material and silyl product, respectively). Uponcompletion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowlyover 60 min so as to maintain the temperature between −20° C. and −10°C. (note: strongly exothermic), followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h, at which point it was an off-white thick suspension. TLC indicated acomplete conversion to the triazole product (EtOAc, Rf 0.87 to 0.75 withthe product spot glowing in long wavelength UV light). The reaction wascooled to −15° C. and water (5 L) was slowly added at a rate to maintainthe temperature below +10° C. in order to quench the reaction and toform a homogenous solution. (Caution: this reaction is initially verystrongly exothermic). Approximately one-half of the reaction volume (22L) was transferred by air pump to another vessel, diluted with EtOAc (12L) and extracted with water (2×8 L). The second half of the reaction wastreated in the same way. The combined aqueous layers were back-extractedwith EtOAc (8 L) The organic layers were combined and concentrated in a20 L rotary evaporator to an oily foam. The foam was coevaporated withanhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be usedinstead of anhydrous acetonitrile if dried to a hard foam). The residuewas dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750mL) was added. A homogenous solution formed in a few minutes and thereaction was allowed to stand overnight

TLC indicated a complete reaction (CH2Cl2-acetone-MeOH, 20:5:3, Rf0.51). The reaction solution was concentrated on a rotary evaporator toa dense foam and slowly redissolved in warm CH₂Cl₂ (4 L, 40° C.) andtransferred to a 20 L glass extraction vessel equipped with aair-powered stirrer. The organic layer was extracted with water (2×6 L)to remove the triazole by-product. (Note: In the first extraction anemulsion formed which took about 2 h to resolve). The water layer wasback-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water(3 L). The combined organic layer was concentrated in 2×20 L flasks to agum and then recrystallized from EtOAc seeded with crystalline product.After sitting overnight, the first crop was collected on a 25 cm CoorsBuchner funnel and washed repeatedly with EtOAc until a whitefree-flowing powder was left (about 3×3 L). The filtrate wasconcentrated to an oil recrystallized from EtOAc, and collected asabove. The solid was air-dried in pans for 48 h, then further dried in avacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a brightwhite, dense solid (86%). An HPLC analysis indicated both crops to be99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAcremained.

Preparation of5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidinepenultimate intermediate

Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine(1000 g, 1.62 mol) was suspended in anhydrous DMF (3 kg) at ambienttemperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3g, 1.94 mol) was added in one portion. The solution clarified after 5hours and was stirred for 16 h. HPLC indicated 0.45% starting materialremained (as well as 0.32% N4,3′-O-bis Benzoyl). An additional amount ofbenzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLCindicated no starting material was present. TEA (450 mL, 3.24 mol) andtoluene (6 L) were added with stirring for 1 minute. The solution waswashed with water (4×4 L), and brine (2×4 L). The organic layer waspartially evaporated on a 20 L rotary evaporator to remove 4 L oftoluene and traces of water. HPLC indicated that the bis benzoyl sideproduct was present as a 6% impurity. The residue was diluted withtoluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride(60% in oil, 70 g, 1.75 mol) was added in one portion with stirring atambient temperature over 1 h. The reaction was quenched by slowly addingthen washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4L), followed by aqueous sodium bicarbonate (2%, 2 L), water (2×4 L) andbrine (4 L). The organic layer was concentrated on a 20 L rotaryevaporator to about 2 L total volume. The residue was purified by silicagel column chromatography (6 L Buchner funnel containing 1.5 kg ofsilica gel wetted with a solution of EtOAc-hexanes-TEA (70:29:1)). Theproduct was eluted with the same solvent (30 L) followed by straightEtOAc (6 L). The fractions containing the product were combined,concentrated on a rotary evaporator to a foam and then dried in a vacuumoven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam(98%). HPLC indicated a purity of >99.7%.

Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite)

5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methylcytidine(1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporatedwith toluene (300 ml) at 50° C. under reduced pressure. The mixture wascooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (30 ml) was added, and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (9 L) and hexanes (6 L). The two layers wereseparated and the upper layer was washed with DMF-water (60:40 v/v, 3×3L) and water (3×2 L). The organic layer was dried (Na2SO4), filtered andevaporated. The residue was co-evaporated with acetonitrile (2×2 L)under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40h) to afford 1336 g of an off-white foam (97%).

Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-6-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite)

5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N6-benzoyladenosine(purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene(300 ml) at 50° C. The mixture was cooled to room temperature and2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) andtetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken untilall tetrazole was dissolved, N-methylimidazole (30 ml) was added, andmixture was left at room temperature for 5 hours. TEA (300 ml) wasadded, the mixture was diluted with DMF (1 L) and water (400 ml) andextracted with hexanes (3×3 L). The mixture was diluted with water (1.4L) and extracted with the mixture of toluene (9 L) and hexanes (6 L).The two layers were separated and the upper layer was washed withDMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer wasdried (Na₂SO₄), filtered and evaporated to a sticky foam. The residuewas co-evaporated with acetonitrile (2.5 L) under reduced pressure anddried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of anoff-white foam solid (96%).

Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite)

5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-isobutyrlguanosine(purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (200 ml) at 50° C., cooled to roomtemperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g,3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture wasshaken until all tetrazole was dissolved, N-methylimidazole (30 ml) wasadded, and the mixture was left at room temperature for 5 hours. TEA(300 ml) was added, the mixture was diluted with DMF (2 L) and water(600 ml) and extracted with hexanes (3×3 L). The mixture was dilutedwith water (2 L) and extracted with a mixture of toluene (10 L) andhexanes (5 L). The two layers were separated and the upper layer waswashed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and thesolution was washed with water (3×4 L). The organic layer was dried(Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) wasadded, the mixture was shaken for 10 min, and the supernatant liquid wasdecanted. The residue was co-evaporated with acetonitrile (2×2 L) underreduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) toafford 1660 g of an off-white foamy solid (91%).

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylamino-oxy-ethyl) nucleoside amidites2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,EtOAc) indicated a complete reaction. The solution was concentratedunder reduced pressure to a thick oil. This was partitioned betweenCH₂Cl₂ (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L).The organic layer was dried over sodium sulfate, filtered, andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of EtOAc and ethyl 0.20 ether (600 mL) andcooling the solution to −10° C. afforded a white crystalline solid whichwas collected by filtration, washed with ethyl ether (3×200 mL) anddried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%).TLC and NMR spectroscopy were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In the fume hood, ethylene glycol (350 mL, excess) was added cautiouslywith manual stirring to a 2 L stainless steel pressure reactorcontaining borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution:evolves hydrogen gas).5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambienttemperature and opened. TLC (EtOAc, Rf 0.67 for desired product and Rf0.82 for ara-T side product) indicated about 70% conversion to theproduct. The solution was concentrated under reduced pressure (10 to 1mm Hg) in a warm water bath (40-100° C.) with the more extremeconditions used to remove the ethylene glycol. (Alternatively, once theTHF has evaporated the solution can be diluted with water and theproduct extracted into EtOAc). The residue was purified by columnchromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). Theappropriate fractions were combined, evaporated and dried to afford 84 gof a white crisp foam (50%), contaminated starting material (17.4 g, 12%recovery) and pure reusable starting material (20 g, 13% recovery). TLCand NMR spectroscopy were consistent with 99% pure product.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P2O5 underhigh vacuum for two days at 40° C. The reaction mixture was flushed withargon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle).Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture with the rate of addition maintained such that theresulting deep red coloration is just discharged before adding the nextdrop. The reaction mixture was stirred for 4 hrs., after which time TLC(EtOAc:hexane, 60:40) indicated that the reaction was complete. Thesolvent was evaporated in vacuo and the residue purified by flash columnchromatography (eluted with 60:40 EtOAc:hexane), to yield2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%) upon rotary evaporation.

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate washed with ice coldCH₂Cl₂, and the combined organic phase was washed with water and brineand dried (anhydrous Na₂SO₄). The solution was filtered and evaporatedto afford 2′-β-(aminooxyethyl) thymidine, which was then dissolved inMeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) wasadded and the resulting mixture was stirred for 1 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography to yield5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%) upon rotary evaporation.

5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C.under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded and the reaction mixture was stirred. After 10 minutes thereaction was warmed to room temperature and stirred for 2 h. while theprogress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂).Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product wasextracted with EtOAc (2×20 mL). The organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated to dryness. This entireprocedure was repeated with the resulting residue, with the exceptionthat formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolutionof the residue in the PPTS/MeOH solution. After the extraction andevaporation, the residue was purified by flash column chromatography and(eluted with 5% MeOH in CH₂Cl₂) to afford5′-O-tert-butyldiphenylsilyl-2′-O—[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%) upon rotary evaporation.

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to5′-O-tert-butyldiphenylsilyl-2′-O—[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removedunder vacuum and the residue purified by flash column chromatography(eluted with 10% MeOH in CH₂Cl₂) to afford2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotaryevaporation of the solvent.

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P2O5 under high vacuum overnight at 40° C., co-evaporatedwith anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to thepyridine solution and the reaction mixture was stirred at roomtemperature until all of the starting material had reacted. Pyridine wasremoved under vacuum and the residue was purified by columnchromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops ofpyridine) to yield5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%)upon rotary evaporation.

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylaminetetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried overP₂O₅ under high vacuum overnight at 40° C. This was dissolved inanhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 h under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, thenthe residue was dissolved in EtOAc (70 mL) and washed with 5% aqueousNaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue obtained was purified by columnchromatography (EtOAc as eluent) to afford5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%) upon rotary evaporation.

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside may bephosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethyl-amino-ethoxy-ethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowlyadded to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves as thesolid dissolves). O2-,2′-anhydro-5-methyhuidine (1.2 g, 5 mmol), andsodium bicarbonate (2.5 mg) were added and the bomb was sealed, placedin an oil bath and heated to 155° C. for 26 h. then cooled to roomtemperature. The crude solution was concentrated, the residue wasdiluted with water (200 mL) and extracted with hexanes (200 mL). Theproduct was extracted from the aqueous layer with EtOAc (3×200 mL) andthe combined organic layers were washed once with water, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel column chromatography (eluted with 5:100:2MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combinedand evaporated to afford the product as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethyl-aminoethoxy)-ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethyl-amino-ethoxy)ethyl)]-5-methyl uridine inanhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytritylchloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h.The reaction mixture was poured into water (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturatedNaHCO₃ solution, followed by saturated NaCl solution, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel column chromatography (eluted with 5:100:1MeOH/CH₂Cl₂/TEA) to afford the product.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) were added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylamino-ethoxy)-ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel columnchromatography with EtOAc as the eluent to afford the title compound.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides:

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides:

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligo-nucleosides,as well as mixed backbone compounds having, for instance, alternatingMMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 4 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 394, asabove. Oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNAportion and 5′-dimethoxy-trityl-2′-O-methyl-3′-O-phosphoramidite for 5′and 3′ wings. The standard synthesis cycle is modified by incorporatingcoupling steps with increased reaction times for the5′-dimethoxy-trityl-2′-O-methyl-3′-O-phosphoramidite. The fullyprotected oligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2-O-methyl chimeric oligonucleotide, with the substitutionof 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphoro-thioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]Chimeric Oligo-nucleo-tides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleo-tides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Design and Screening of Duplexed Compounds Targeting ForkheadBox O1A

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target forkhead box O1A. Thenucleobase sequence of the antisense strand of the duplex comprises atleast an 8-nucleobase portion of an oligonucleotide in Table 1. The endsof the strands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO:169) and having a two-nucleobase overhangof deoxythymidine(dT) would have the following structure:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate forkhead box O1A expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 7 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32 +/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 8 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 9 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 10 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (ITEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO:1) which is targeted to human H-ras, orISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO:3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-Ha-ras (for ISIS13920) or c-raf (for ISIS15770) mRNAis then utilized as the screening concentration for new oligonucleotidesin subsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 11 Analysis of Oligonucleotide Inhibition of Forkhead Box O1AExpression

Antisense modulation of forkhead box O1A expression can be assayed in avariety of ways known in the art. For example, forkhead box O1A mRNAlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+ mRNA. The preferred method of RNAanalysis of the present invention is the use of total cellular RNA asdescribed in other examples herein. Methods of RNA isolation are taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

Protein levels of forkhead box O1A can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to forkhead box O1A can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional antibody generation methods. Methods for preparation ofpolyclonal antisera are taught in, for example, Ausubel, F. M. et al.,(Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9,John Wiley & Sons, Inc., 1997). Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997).

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., (Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998).Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al.,(Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22,John Wiley & Sons, Inc., 1991).

Example 12 Design of Phenotypic Assays and In Vivo Studies for the Useof Forkhead Box O1A Inhibitors

Phenotypic Assays

Once forkhead box O1A inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of forkhead box O1A in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withforkhead box O1A inhibitors identified from the in vitro studies as wellas control compounds at optimal concentrations which are determined bythe methods described above. At the end of the treatment period, treatedand untreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the forkhead box O1A inhibitors.Hallmark genes, or those genes suspected to be associated with aspecific disease state, condition, or phenotype, are measured in bothtreated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study. To account for the psychologicaleffects of receiving treatments, volunteers are randomly given placeboor forkhead box O1A inhibitor. Furthermore, to prevent the doctors frombeing biased in treatments, they are not informed as to whether themedication they are administering is a forkhead box O1A inhibitor or aplacebo. Using this randomization approach, each volunteer has the samechance of being given either the new treatment or the placebo.

Volunteers receive either the forkhead box O1A inhibitor or placebo foreight week period with biological parameters associated with theindicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingforkhead box O1A or forkhead box O1A protein levels in body fluids,tissues or organs compared to pre-treatment levels. Other measurementsinclude, but are not limited to, indices of the disease state orcondition being treated, body weight, blood pressure, serum titers ofpharmacologic indicators of disease or toxicity as well as ADME(absorption, distribution, metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and forkhead box O1A inhibitor treatment. Ingeneral, the volunteers treated with placebo have little or no responseto treatment, whereas the volunteers treated with the forkhead box O1Ainhibitor show positive trends in their disease state or condition indexat the conclusion of the study.

Example 13 Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation aretaught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993). Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70 μC, was added to each well, the plate wasincubated on a 90 μl hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 14 Total RNA Isolation

Total RNA was isolated using an RNEASY96™ kit and buffers purchased fromQiagen Inc. (Valencia, Calif.) following the manufacturer's recommendedprocedures. Briefly, for cells grown on 96-well plates, growth mediumwas removed from the cells and each well was washed with 200 μL coldPBS. 150 μL Buffer RLT was added to each well and the plate vigorouslyagitated for 20 seconds. 150 μL of 70% ethanol was then added to eachwell and the contents mixed by pipetting three times up and down. Thesamples were then transferred to the RNEASY 96™ well plate attached to aQIAVAC™ manifold fitted with a waste collection tray and attached to avacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 wasadded to each well of the RNEASY 96™ plate and incubated for 15 minutesand the vacuum was again applied for 1 minute. An additional 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumwas applied for 2 minutes. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 90seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 3 minutes. The plate was then removed from theQIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 170μL water into each well, incubating 1 minute, and then applying thevacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 15 Real-Time Quantitative PCR Analysis of Forkhead Box O1A mRNALevels

Quantitation of forkhead box O1A mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCRin which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems,Foster City, Calif., Operon Technologies Inc., Alameda, Calif. orIntegrated DNA Technologies Inc., Coralville, Iowa) is attached to the5′ end of the probe and a quencher dye (e.g., TAMRA, obtained fromeither PE-Applied Biosystems, Foster City, Calif., Operon TechnologiesInc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville,Iowa) is attached to the 3′ end of the probe. When the probe and dyesare intact, reporter dye emission is quenched by the proximity of the 3′quencher dye. During amplification, annealing of the probe to the targetsequence creates a substrate that can be cleaved by the 5′-exonucleaseactivity of Taq polymerase. During the extension phase of the PCRamplification cycle, cleavage of the probe by Taq polymerase releasesthe reporter dye from the remainder of the probe (and hence from thequencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™7700 Sequence Detection System. In each assay, a series of parallelreactions containing serial dilutions of mRNA from untreated controlsamples generates a standard curve that is used to quantitate thepercent inhibition after antisense oligonucleotide treatment of testsamples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer (—MgCl2), 6.6 mM MgCl₂, 375 μM each of dATP, dCTP, dCTPand dGTP, 375 nM each of forward primer and reverse primer, 125 nM ofprobe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLVreverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30μL total RNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent from Molecular Probes. Methods of RNA quantification byRiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry,1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nmand emission at 520 nm.

Probes and primers to human forkhead box O1A were designed to hybridizeto a human forkhead box O1A sequence, using published sequenceinformation (GenBank accession number NM 002015.2, incorporated hereinas SEQ ID NO:4). For human forkhead box O1A the PCR primers were:

(SEQ ID NO: 5) forward primer: GCAATCCCGAAAACATGGAA (SEQ ID NO: 6)reverse primer: CAGGTGAGGACTGGGTCGAAand the PCR probe was:

FAM-TGGATAATCTCAACCTTCTCTCATCACCAACATC-TAMRA (SEQ ID NO:7)

where FAM is the fluorescent dye and TAMRA is the quencher dye. Forhuman GAPDH the PCR primers were:

(SEQ ID NO: 8) forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 9)reverse primer: GAAGATGGTGATGGGATTTCand the PCR probe was:

5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO:10) where JOE is thefluorescent reporter dye and TAMRA is the quencher dye.

Probes and primers to mouse forkhead box O1A were designed to hybridizeto a mouse forkhead box O1A sequence, using published sequenceinformation (GenBank accession number AJ252157.1, incorporated herein asSEQ ID NO:11). For mouse forkhead box O1A the PCR primers were:

(SEQ ID NO: 12) forward primer: CAAAGTACACATACGGCCAATCC (SEQ ID NO: 13)reverse primer: CGTAACTTGATTTGCTGTCCTGAAand the PCR probe was:

(SEQ ID NO: 14) FAM-TGAGCCCTTTGCCCCAGATGCCTAT-TAMRAwhere FAM is the fluorescent reporter dye and TAMRA is the quencher dye.For mouse GAPDH the PCR primers were:

(SEQ ID NO: 15) forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 16)reverse primer: GGGTCTCGCTCCTGGAAGATand the PCR probe was:

(SEQ ID NO: 17) 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC- TAMRA 3′where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 16 Northern Blot Analysis of Forkhead Box O1A mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human forkhead box O1A, a human forkhead box O1A specificprobe was prepared by PCR using the forward primer GCAATCCCGAAAACATGGAA(SEQ ID NO:5) and the reverse primer CAGGTGAGGACTGGGTCGAA (SEQ ID NO:6).To normalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

To detect mouse forkhead box O1A, a mouse forkhead box O1A specificprobe was prepared by PCR using the forward primerCAAAGTACACATACGGCCAATCC (SEQ ID NO:12) and the reverse primerCGTAACTTGATTTGCTGTCCTGAA (SEQ ID NO:13). To normalize for variations inloading and transfer efficiency membranes were stripped and probed formouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 17 Modulation of Human Forkhead Box O1A Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human forkhead box O1ARNA, using published sequences (GenBank accession number NM_(—)002015.2,incorporated herein as SEQ ID NO:4). The oligonucleotides are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human forkhead box O1A mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom two experiments in which T-24 cells were treated with the antisenseoligonucleotides of the present invention. The positive control for eachdatapoint is identified in the table by sequence ID number. If present,“N.D.” indicates “no data”.

TABLE 1 Inhibition of human forkhead box O1A mRNA levels by chimeric phosphorothioate oligonucleotides having  2′-MOE wings and a deoxy gap TARGET SEQ CONTROL ISIS SEQ ID TARGET % IDSEQ ID # REGION NO SITE SEQUENCE INHIB NO NO 188750 5′UTR 4    4tgcctgttgaatgtggcggc 65 18 2 188755 Coding 4  457 ccggcctgggcagcggccag79 19 2 188757 Coding 4  701 ccctggaagtccccgcacag 77 20 2 188759 Coding4  878 ttggtgatgaggtcggcgta 91 21 2 188761 Coding 4  889tctcgatggccttggtgatg 80 22 2 188763 Coding 4  959 ttgaagtagggcacgctctt72 23 2 188765 Coding 4 1092 ctctggattgagcatccacc 61 24 2 188766 Coding4 1155 aaatttactgttgttgtcca 63 25 2 188767 Coding 4 1161cttagcaaatttactgttgt 51 26 2 188769 Coding 4 1205 ccagactggagagatgcttt69 27 2 188771 Coding 4 1304 aatgtactccagttatcaaa 58 28 2 188773 Coding4 1510 agagaaggttgagattatcc 75 29 2 188774 Coding 4 1664gggctcatgctggattggcc 63 30 2 188776 Coding 4 1782 gggaggagagtcagaagtca 0 31 2 188778 Coding 4 1904 tgagatgcctggctgccata 87 32 2 188780 Coding4 1916 atcattttgttatgagatgc 75 33 2 188781 Coding 4 2091cagggcactcatctgcatgg 74 34 2 188782 Coding 4 2126 tagccattgcagctgctcac84 35 2 188783 Coding 4 2162 gggagcttctcctggtggag 27 36 2 188785 Coding4 2176 catccaagtcacttgggagc 73 37 2 188787 Coding 4 2231aggtcattccgaatgatgga 61 38 2 188789 Coding 4 2246 gtatctccatccatgaggtc55 39 2 188790 Coding 4 2285 ctttggttgggcaacacatt 63 40 2 188791 Coding4 2290 ggaagctttggttgggcaac 60 41 2 188793 Coding 4 2302tgacactgtgtgggaagctt 85 42 2 188795 3′UTR 4 2384 tgctgtcagacaatctgaag 6243 2 188797 3′UTR 4 2575 ggcacagtccttatctacag 83 44 2 188799 3′UTR 42605 ttggcacttcattgtaatga 76 45 2 188801 3′UTR 4 2617ggtgtagtgagtttggcact 72 46 2 188803 3′UTR 4 2753 aaagagtataaactttcctt 6447 2 188805 3′UTR 4 3046 tgtacaaatttgcaaataac 76 48 2 188807 3′UTR 43079 catttatctggaaattagaa 44 49 2 188809 3′UTR 4 3163tagactctagttttaagaaa 34 50 2 188811 3′UTR 4 3174 atgtaacaaagtagactcta 7651 2 188813 3′UTR 4 3265 taaaattagtactaatccag 73 52 2 188815 3′UTR 43467 tgaaatttctttaaaataca  0 53 2 188820 3′UTR 4 4919aatcaaacaaggctgcatag 57 54 2

As shown in Table 1, SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 51, 52 and 54 demonstrated at least 50% inhibition of human forkheadbox O1A expression in this assay. SEQ ID NOs 21, 32, and 42 areparticularly effective at inhibiting forkhead box O1A expression. Thetarget sites to which these sequences are complementary are hereinreferred to as “target regions” and are suitable sites for targeting bycompounds of the present invention. These target regions are shown inTable 3. The sequences represent the reverse complement of the compoundsshown in Table 1. “Target site” indicates the first (5′-most) nucleotidenumber of the corresponding target nucleic acid. Also shown in Table 3is the species in which each of the preferred target regions was found.

Example 18 Modulation of Mouse Forkhead Box O1A Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseforkhead box O1A RNA, using published sequences (GenBank accessionnumber AJ252157.1, incorporated herein as SEQ ID NO:11, GenBankaccession number BE198396.1, incorporated herein as SEQ ID NO:55, andthe complement of GenBank accession number AA959612.1, incorporatedherein as SEQ ID NO:56). The oligonucleotides are shown in Table 2.“Target site” indicates the first (5′-most) nucleotide number on theparticular target sequence to which the oligonucleotide binds. Allcompounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on mouse forkhead box O1A mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom two experiments in which b.END cells were treated with theantisense oligonucleotides of the present invention. The positivecontrol for each datapoint is identified in the table by sequence IDnumber. If present, “N.D.” indicates “no data”.

TABLE 2 Inhibition of mouse forkhead box O1A mRNA levels bychimeric phosphorothioate oligonucleotides having2′-MOE wings and a deoxy gap TARGET SEQ CONTROL ISIS SEQ ID TARGET % IDSEQ ID # REGION NO SITE SEQUENCE INHIB NO NO 188749 5′UTR 11    9ctcccgtcttacgggatctg 18 57 1 188751 5′UTR 11   61 cgctgctgcctgttgaatgt29 58 1 188752 5′UTR 11  223 tgtctaaggagcctccgagt  46 59 1 188753 5′UTR11  384 cagagaagtaccgggagacg  39 60 1 188754 Start 11  419cttcggccatggtgcccccg 63 61 1 Codon 188756 Coding 11  506taaactccggcctgggcagc 42 62 1 188758 Coding 11  889 caggttgccccacgcgttgc67 63 1 188760 Coding 11  917 tggccttggtgatgaggtcg 17 64 1 188762 Coding11  946 ggtgagcctcttctcggctg 54 65 1 188764 Coding 11 1096tccagttccttcattctgca 77 66 1 188768 Coding 11 1201 tcggctcttagcaaatttac32 67 1 188770 Coding 11 1332 ctccagttatcaaagtcatc 45 68 1 188772 Coding11 1487 acagactgggcagcgtagac 36 69 1 188775 Coding 11 1704ggcaaagggctcatgctgga 87 70 1 188777 Coding 11 1908 accgaattagggcccatcat50 71 1 188779 Coding 11 1944 ttgttatgagatgcctggct 35 72 1 188784 Coding11 2202 tcacttgggagcttctcctg 50 73 1 188786 Coding 11 2216acatgccatccaagtcactt 32 74 1 188788 Coding 11 2271 tccatgaggtcattccgaat59 75 1 188792 Coding 11 2330 tgtgtgggaagctttggttg 35 76 1 188794 Stop11 2377 cactaactcttagcctgaca 57 77 1 Codon 188796 3′UTR 11 2424agttcctactgtcagacaat 43 78 1 188798 3′UTR 11 2611 ccaatggcacagtccttatc56 79 1 188800 3′UTR 11 2640 gtgagtttggcacttcattg 50 80 1 188802 3′UTR11 2769 ataaactttccttggaccaa 72 81 1 188804 3′UTR 11 3011agacctgtacaaagctggca 69 82 1 188806 3′UTR 11 3090 tctggaaattagaaccattt38 83 1 188808 3′UTR 11 3169 ctagttttaagaaaacatta 18 84 1 188810 3′UTR11 3181 acaaagtagactctagtttt 34 85 1 188812 3′UTR 11 3272tagtactaatccagttagaa 34 86 1 188814 3′UTR 11 3363 tgcaagtactaattacaatg33 87 1 188816 3′UTR 11 3505 tggtgctatgcgctgtacac 54 88 1 188817 3′UTR11 3905 agctggctggtttccaagtt 58 89 1 188818 3′UTR 11 4141ggccttctcataaaggcaaa 47 90 1 188819 3′UTR 11 4691 agttcactgtgccccagaca68 91 1 188821 3′UTR 11 4923 gaaacaatacatctttatat 26 92 1 188822 3′UTR55  131 gactcagtttgtccaagcag 68 93 1 188823 3′UTR 55  413gtttggtttgcataaagcac 51 94 1 188824 3′UTR 55  423 gggccaggctgtttggtttg 2 95 1 188825 3′UTR 56  271 ggcacttctcagatagcaat 67 96 1 188826 3′UTR56  351 gaggatttatgtacatttat  0 97 1As shown in Table 2, SEQ ID NOs 59, 60, 61, 62, 63, 65, 66, 68, 69, 70,71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 88, 89, 90, 91, 93, 94and 96 demonstrated at least 35% inhibition of mouse forkhead box O1Aexpression in this experiment. Compounds having SEQ ID Nos 66 70, and 81(compounds 188764, 188775, and 188802) are particularly effective atinhibiting forkhead box O1A expression. The target sites to which thesepreferred sequences are complementary are herein referred to as “targetregions” and are suitable sites for targeting by compounds of thepresent invention. These target regions are shown in Table 3. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Table 1. “Target site” indicates the first (5′-most)nucleotide number of the corresponding target nucleic acid. Also shownin Table 3 is the species in which each of the target regions was found.

TABLE 3 Sequence and position of preferred target regions identified in forkhead box O1A. TARGET REV SEQ SITE SEQ ID TARGETCOMP OF ID ID NO SITE SEQUENCE SEQ ID ACTIVE IN NO 104287  4   4gccgccacattcaacaggca 18 H. sapiens  98 104292  4  457ctggccgctgcccaggccgg 19 H. sapiens  99 104294  4  701ctgtgcggggacttccaggg 20 H. sapiens 100 104296  4  878tacgccgacctcatcaccaa 21 H. sapiens 101 104298  4  889catcaccaaggccatcgaga 22 H. sapiens 102 104300  4  959aagagcgtgccctacttcaa 23 H. sapiens 103 104302  4 1092ggtggatgctcaatccagag 24 H. sapiens 104 104303  4 1155tggacaacaacagtaaattt 25 H. sapiens 105 104304  4 1161acaacagtaaatttgctaag 26 H. sapiens 106 104306  4 1205aaagcatctctccagtctgg 27 H. sapiens 107 104308  4 1304tttgataactggagtacatt 28 H. sapiens 108 104310  4 1510ggataatctcaaccttctct 29 H. sapiens 109 104311  4 1664ggccaatccagcatgagccc 30 H. sapiens 110 104315  4 1904tatggcagccaggcatctca 32 H. sapiens 111 104317  4 1916gcatctcataacaaaatgat 33 H. sapiens 112 104318  4 2091ccatgcagatgagtgccctg 34 H. sapiens 113 104319  4 2126gtgagcagctgcaatggcta 35 H. sapiens 114 104322  4 2176gctcccaagtgacttggatg 37 H. sapiens 115 104324  4 2231tccatcattcggaatgacct 38 H. sapiens 116 104326  4 2246gacctcatggatggagatac 39 H. sapiens 117 104327  4 2285aatgtgttgcccaaccaaag 40 H. sapiens 118 104328  4 2290gttgcccaaccaaagcttcc 41 H. sapiens 119 104330  4 2302aagcttcccacacagtgtca 42 H. sapiens 120 104332  4 2384cttcagattgtctgacagca 43 H. sapiens 121 104334  4 2575ctgtagataaggactgtgcc 44 H. sapiens 122 104336  4 2605tcattacaatgaagtgccaa 45 H. sapiens 123 104338  4 2617agtgccaaactcactacacc 46 H. sapiens 124 104340  4 2753aaggaaagtttatactcttt 47 H. sapiens 125 104342  4 3046gttatttgcaaatttgtaca 48 H. sapiens 126 104348  4 3174tagagtctactttgttacat 51 H. sapiens 127 104350  4 3265ctggattagtactaatttta 52 H. sapiens 128 104357  4 4919ctatgcagccttgtttgatt 54 H. sapiens 129 104289 11  223actcggaggctccttagaca 59 M. musculus 130 104290 11  384cgtctcccggtacttctctg 60 M. musculus 131 104291 11  419cgggggcaccatggccgaag 61 M. musculus 132 104293 11  506gctgcccaggccggagttta 62 M. musculus 133 104295 11  889gcaacgcgtggggcaacctg 63 M. musculus 134 104299 11  946cagccgagaagaggctcacc 65 M. musculus 135 104301 11 1096tgcagaatgaaggaactgga 66 M. musculus 136 104307 11 1332gatgactttgataactggag 68 M. musculus 137 104309 11 1487gtctacgctgcccagtctgt 69 M. musculus 138 104312 11 1704tccagcatgagccctttgcc 70 M. musculus 139 104314 11 1908atgatgggccctaattcggt 71 M. musculus 140 104316 11 1944agccaggcatctcataacaa 72 M. musculus 141 104321 11 2202caggagaagctcccaagtga 73 M. musculus 142 104325 11 2271attcggaatgacctcatgga 75 M. musculus 143 104329 11 2330caaccaaagcttcccacaca 76 M. musculus 144 104331 11 2377tgtcaggctaagagttagtg 77 M. musculus 145 104333 11 2424attgtctgacagcaggaact 78 M. musculus 146 104335 11 2611gataaggactgtgccattgg 79 M. musculus 147 104337 11 2640caatgaagtgccaaactcac 80 M. musculus 148 104339 11 2769ttggtccaaggaaagtttat 81 M. musculus 149 104341 11 3011tgccagctttgtacaggtct 82 M. musculus 150 104343 11 3090aaatggttctaatttccaga 83 M. musculus 151 104353 11 3505gtgtacagcgcatagcacca 88 M. musculus 152 104354 11 3905aacttggaaaccagccagct 89 M. musculus 153 104355 11 4141tttgcctttatgagaaggcc 90 M. musculus 154 104356 11 4691tgtctggggcacagtgaact 91 M. musculus 155 104359 55  131ctgcttggacaaactgagtc 93 M. musculus 156 104360 55  413gtgctttatgcaaaccaaac 94 M. musculus 157 104362 56  271attgctatctgagaagtgcc 96 M. musculus 158As these “target regions” have been found by experimentation to be opento, and accessible for, hybridization with the compounds of the presentinvention, one of skill in the art will recognize or be able toascertain, using no more than routine experimentation, furtherembodiments of the invention that encompass other compounds thatspecifically hybridize to these sites and consequently inhibit theexpression of forkhead box O1A.

In one embodiment, the “target region” may be employed in screeningcandidate antisense compounds. “Candidate” compounds are those thatinhibit the expression of a nucleic acid molecule encoding forkhead boxO1A and which comprise at least an 8-nucleobase portion which iscomplementary to a target region. The method comprises the steps ofcontacting a target region of a nucleic acid molecule encoding forkheadbox O1A with one or more candidate compounds, and selecting for one ormore candidate antisense compounds which inhibit the expression of anucleic acid molecule encoding forkhead box O1A. Once it is shown thatthe candidate compound or compounds are capable of inhibiting theexpression of a nucleic acid molecule encoding forkhead box O1A, thecandidate compound may be employed as a compound in accordance with thepresent invention.

According to the present invention, compounds include ribozymes,external guide sequence (EGS) oligonucleotides (oligozymes), and othershort catalytic RNAs or catalytic oligonucleotides which hybridize tothe target nucleic acid and modulate its expression.

Example 19 Western Blot Analysis of Forkhead Box O1A Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to forkhead box O1A isused, with a radiolabeled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 20 In Vitro Modulation of Forkhead Box O1A

Using the methods described in Example 15, a dose-dependent reduction offorkhead box O1A mRNA and protein was achieved with compound 188764 inprimary murine hapatocytes.

In another experiment, compounds 188764, 188781, and 188802 resulted ina dose-dependent reduction in forkhead box O1A mRNA (expressed as % notreatment) (see Table 4).

TABLE 4 Compound Concentration 188764 Compound 188781 Compound 188802  1nM 65% 62 60  10 nM 38 58 62  50 nM 18 48 55 100 nM  8 23 45 150 nM 1023 30

Example 21 Four Week Lean Mouse Model

C57B16 (normal lean) mice were used in this 4 week lean mouse screen.These mice are commercially available through any vendor, such asJackson Laboratories or Charles River, and are considered to be normal.The term “lean” is used to contrast these mice to obese db/db mice.Several compounds including 188775, 188793, 188764, 188802, and 141923(a scramble sequence used as a control—it does not bind to forkhead boxO1A) were administered to C57B16 mice to determine the effect onforkhead expression. Forkhead box O1A mRNA expression was found to bereduced in both liver and fat using the procedures described in theearlier examples. Compound 188802, 188793, and 188764 resulted in thegreatest amount of reduction of forkhead box O1A mRNA expression.

In another experiment, administering compound 188764 resulted in adecrease in liver forkhead box O1A mRNA (40%) and protein (45%) (samestudy design as the previous one 4 weeks treatment).

In another experiment were C57B16 mice were treated for 2 weeks,compounds 188764, 188781, and 188802 resulted in a decrease in forkheadbox O1A mRNA in liver (results expressed as % of saline control)(compound 188764=50%; compound 188781=79%; compound 188802=70%).

In another experiment, C57B16 lean mice were treated with Compounds188764 (12.5 and 25 mg/kg×2 weekly), or 141923 (25 mg/kg×2 weekly), orsaline for 4 weeks. Plasma glucose and lipid levels were measured everyother week (at 0, 2, and 4 weeks). At week 3, mice were fasted for 12hours and a glucose tolerance test was performed (glucose tolerance testmeasures the body's ability to metabolize glucose). At the 4^(th) week,the mice were sacrificed and liver, fat, muscle, kidneys, pancreas, andbone marrow were collected. Compound 188764 significantly improved theglucose tolerance test at 12.5 and 25 (2) mg/kg/week (FIG. 1) anddecreased insulin levels by more than 50% at the highest dose (FIG. 2).

Example 22 db/db Mouse Model

The db/db diabetic mouse model was used in this screen to furtherexamine compound 188764 in regard to fed and fasted blood glucose andmonitored at 0, 2, and 4 weeks. The db/db mice are commerciallyavailable from The Jackson Laboratory JAX® GEMM® Strain—SpontaneousMutation Congenic Mice, and are homozygous for the diabetes spontaneousmutation (Lepr^(db)). These mice become identifiably obese around 3 to 4weeks of age. db/db mice at 9 weeks of age were injected twice a weekwith saline or compound 188764 for 4 weeks. Blood glucose was measuredevery other week. In one experiment, mice were fasted for 10, 12, and 16hours and blood glucose was measured at the end of the fourth week. Fedplasma glucose levels were improved with compound 188764 and fastedblood glucose levels were also improved (results expressed as bloodglucose mg/dl) (saline=about 220 at 10 hour fast, about 290 at 12 hoursfast, and about 160 at 16 hours fast; compound 188764=about 170 at 10hour fast, about 125 at 12 hours fast, and about 100 at 16 hours fast).Forkhead box O1A mRNA expression was reduced in both liver and fat. Fedblood glucose was consistently decreased by compound 188764 treatment atthe highest dose by 100 mg/dl compared to fed blood glucose from salineand 141923 treated groups.

In another experiment, compound 188764 decreased blood glucose in thefasted and fed state.

In another experiment, compound 188764 significantly reduced bloodglucose levels after a 16 hour fast in 9-12 weeks db/db mice (see, FIG.3).

In another experiment, compound 188764 resulted in a decrease inforkhead box O1A mRNA and protein in liver and fat between 60-80%.

Example 23 High Fat Diet (HFD) Mouse Model

C57B16 mice fed with a high fat diet (high fat diet (HFD) induceddiabetes after about 12 weeks of feeding) was used in this screen tofurther examine compound 188764 in regard to a glucose tolerance testand insulin tolerance test (insulin tolerance test assesses insulinsensitivity/presence of insulin resistance, as described in Black,Metabolism, 1998, 47, 1354-1359). C57B16 mice show high susceptibilityto development of moderate obesity, hyper-glycemia, and insulinresistance when fed a high fat diet. In general, HFD fed mice were at 3weeks of age started with HFD for 12 weeks and injected twice a weekwith saline or compounds for 4 weeks. Blood glucose was measured everyother week, and plasma glucose and lipid levels were measured everyother even week. At week 3, a glucose tolerance test was initiated inwhich 45 mice were fasted for 12 hours and given 2 g/kg glucose. Also atweek 3, an insulin tolerance test was initiated in which another 45 micewere fasted for 4 hours and given 0.5 U/kg insulin. Compound 188764 wasfed at 10, 25, and 50 mg/kg/wk.

Forkhead box O1A mRNA and protein expression was reduced in both liver(between 40-60%) and fat. Compound 188764 decreased plasma glucoselevels in a dose-dependent manner. An improvement in the glucosetolerance test was observed with the feeding regimens of 25 and 50mg/kg/wk of compound 188764 when compared to saline. Compound 188764decreased fed and fasted (4 hour fast) plasma blood glucose levels. Inaddition, a dose response was achieved with compound 188764 with respectto lowering insulin levels (>90% at highest dose). No significantchanges were observed in plasma cholesterol, plasma triglycerides, bodyweight, or ALT/AST (serum activities of alanine and aspartateaminotransferase, respectively) concentrations. Body weights were alsounaffected by compound 188764.

In another experiment, compound 188764 decreased blood glucose in thefed (18%) and fasted states, normalized glucose intolerance, normalizedinsulin levels (decreased by 94%). Compound 188764 (25×2 mg/kg) androsiglitazone (rosiglitazone is a thiazolidinedione, the peroxisomeproliferator-activated receptor-gamma agonists, which work by reducingthe body's resistance to the action of insulin; used as a referencecompound) had similar effects on plasma insulin levels in HFD fed mice(see FIG. 4).

In another experiment, compound 188764 resulted in a decrease inforkhead box O1A mRNA and protein expression in liver and fat between40-80%, and normalized glucose and insulin levels (decreased insulinlevels greater than about 90%).

Exemplary effects of Compound 188764 and rosiglitazone on a glucosetolerance test in high fat fed mice is shown in FIG. 5.

Example 24 Modulation of Human Forkhead O1A Expression

HEPG2 cells were treated with 50 nM compound for 20 hours. mRNA wasanalyzed by RT-PCR (TaqMan 7700), n=3. Results are shown below as theIC₅₀. Modulation of forkhead box O1A expression was correlated tomodulation of glucose-6-phosphate expression for compounds 289841 and289865. Table 5 lists several compounds targeted to human forkhead boxO1A.

TABLE 5 IC₅₀ Human SEQ Compound HepG2 ID # Sequence (nM) NO: 289800tgccccacgcgttgcggcgg 20 159 289865 ggcaacgtgaacaggtccaa 15 160 289875agctgactatgtaacaaagt 24 161 289813 aactgtgatccagggctgtc 25 162 289839cccagggcactcatctgcat 25 163 289841 ctaagcgctcaatgaacatg 25 164 289866tagcagattgataacaggct 25 165 289826 ggctgggtgaattcaaactg 30 166 188793tgacactgtgtgggaagctt 30  42 289834 catgaccgaattagggccca 35 167 289842cattccgaatgatggattcc 40 168 188790 ctttggttgggcaacacatt 38  40 188812tagtactaatccagttagaa 25  86

Example 25 Modulation Of Monkey Forkhead O1A Expression

Monkey primary hepatocytes were treated with 50 nM of antisensecompounds for 20 hours. Levels of mRNA were analyzed by RT-PCR (TaqMan7700). The results are shown in Table 6.

TABLE 6 Monkey SEQ Compound Hepatocyte ID # Sequence IC₅₀ (nM) NO:327652 gctttggttgggcaacacat 20 172 327612 ccgcttctccgccgagctct 18 173327643 ccgccagggcactcatctgc 15 174 327658 tgatctacagttcctgct 19 175327623 catagaatgcacatcccctt 24 176

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

What is claimed is:
 1. A compound comprising a modified oligonucleotideconsisting of 8 to 80 linked nucleosides and having a nucleobasesequence with at least 80% complementarity to a nucleic acid moleculeencoding forkhead box O1A (SEQ ID NO: 4) and comprising at least 8contiguous nucleobases of a nucleobase sequence recited in any one ofSEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33,34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 51, 52, 54, 86,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 173, 174, 175, and176, wherein the modified oligonucleotide comprises at least onemodification selected from a modified internucleoside linkage, amodified sugar, and a modified nucleobase.
 2. The compound of claim 1,wherein the modified oligonucleotide consists of 8 to 50 linkednucleosides.
 3. The compound of claim 1, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides.
 4. The compoundof claim 1, wherein the modified oligonucleotide is a single-strandedoligonucleotide.
 5. The compound of claim 1, wherein the modifiedoligonucleotide is a DNA oligonucleotide.
 6. The compound of claim 1,wherein the modified oligonucleotide is an RNA oligonucleotide.
 7. Thecompound of claim 1, wherein at least a portion of the modifiedoligonucleotide hybridizes with RNA to form an oligonucleotide-RNAduplex.
 8. The compound of claim 1, wherein the modified oligonucleotidecomprises at least one modified internucleoside linkage.
 9. The compoundof claim 8, wherein the modified internucleoside linkage is aphosphorothioate linkage.
 10. The compound of claim 1, wherein at leastone nucleoside comprises a modified sugar.
 11. The compound of claim 10,wherein the modified sugar is a bicyclic sugar.
 12. The compound ofclaim 10, wherein the modified sugar comprises a 2′-O-methoxyethyl. 13.The compound of claim 1, wherein at least one nucleoside comprises amodified nucleobase.
 14. The compound of claim 13, wherein the modifiednucleobase is a 5-methylcytosine.
 15. A composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 16. The compound of claim 1, wherein the modifiedoligonucleotide consists of 20 linked nucleosides.
 17. The compound ofclaim 1, wherein the nucleobase sequence of the modified oligonucleotideis 99% complementary within SEQ ID NO:
 4. 18. The compound of claim 1,wherein the nucleobase sequence of the modified oligonucleotide is 100%complementary within SEQ ID NO:
 4. 19. The compound of claim 1, whereinthe modified oligonucleotide has a nucleobase sequence comprising anucleobase sequence recited in any one of SEQ ID NOs: 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 51, 52, 54, 86, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 173, 174, 175, and
 176. 20. The compound ofclaim 1, wherein the modified oligonucleotide has a nucleobase sequenceconsisting of a nucleobase sequence recited in any one of SEQ ID NOs:18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 51, 52, 54, 86, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 173, 174, 175, and 176.